The goal of Specific Aim number one is to evaluate the propensity of Diethyl-nitrosamine (initiator) DEN, to form DNA adducts at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in male rats utilizing the granuloma pouch assay. It was thought that this assay would allow for the isolation of the hprt locus from 6-thioguanine resistant clones, and the presence of adducts could be quantified using microcapillary HPLC electrospray ionization tandem mass spectrometry. Based on the critique for this new project the Project Director will follow the advice of the reviewers and not attempt to carry out these studies with male rats because previous studies have demonstrated that DEN/TCDD tumor foci have been identified in female rats only. The described studies will be restricted to female rats and will begin when the tissue culture lab is fully operational. The goal of Specific Aim number two is to test the hypothesis that ovarian hormones exacerbate the promotion of liver tumors by TCDD. This will be evaluated by examining the propensity of 2,3,7,8-Tetrachlorodibenzo-a-dioxin (TCDD) or DEN to form DNA adducts (in hepatic DNA fractions and urine) in control, and ovariectomized rats. We further proposed to assess the quantitate the adducts by microcapillary HPLC electrospray ionization tandem mass spectrometry and thermospray liquid chromatography mass spectrometry. The reviewers expressed concern with respect to the proposed identification of the DEN-TCDD adducts by this methodology because the formation of these adducts has been estimated to occur at a ratio of 1 per 10 (11) nucleotides. They therefore recommended that the mass spectrometry studies be eliminated. Therefore, we have modified Specific Aim number two such that we will focus on the GC-MS studies looking at whether ovariectomy alters tissue distribution of TCDD through altered metabolism/clearance time. We will also examine the possibility of the stimulation of tumor sell growth by estrogens as the cause for the predicted increase in tumor foci formation in control female rats.